Implementation of stable and complex biological systems through recombinase-assisted genome engineering

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Genomic Engineering

Background:

• Plasmid-based systems in engineered biological systems suffer from phenotypic noise due to genetic instability and population dynamics, limiting accuracy and precision.
• A shift towards genomic-level engineering is necessary to minimize uncertainty and achieve robust performance.
• Current methods struggle with the stable implementation of complex heterologous pathways.

Purpose of the Study:

• To introduce and apply recombinase-assisted genome engineering (RAGE) for the stable installment and implementation of complex biological systems.
• To develop a robust Escherichia coli strain for direct ethanol production from brown macroalgae.
• To optimize a 34 kb heterologous pathway for alginate metabolism, including alginate degradation and ethanol production modules.

Main Methods:

• Development of an advanced design principle utilizing RAGE for genomic integration.
• Application of RAGE to engineer Escherichia coli for alginate metabolism and ethanol production.
• Optimization of pathway implementation considering genetic background, integration locus, copy number, and module compatibility.

Main Results:

• RAGE significantly expedited the optimization of a 34 kb heterologous pathway.
• The RAGE-engineered strain achieved a ~40% higher ethanol titre compared to its plasmid-based counterpart.
• After 50 generations, the engineered strain demonstrated substantial improvements: ~330% in titre and ~1,200% in productivity.

Conclusions:

• RAGE provides a robust and stable platform for engineering complex biological systems at the genomic level.
• Genomic engineering via RAGE overcomes limitations of plasmid-based systems, leading to significantly enhanced performance.
• This approach enables efficient direct ethanol production from brown macroalgae, showcasing potential for industrial biotechnology.